Cancer is driven by genetic or epigenetic alterations that directly affect the function of critical oncogenes and tumor suppressors. Together, these pivotal proteins underlie fundamental mechanisms of cancer development. While there have been significant efforts towards elaborating canonical oncogene/tumor suppressor circuits, a new era of understanding the epigenetic basis of cancer is beginning. How these new epigenetic changes in cancer cells integrate oncogene/tumor suppressor functions is not known. The central concept of this application is that the Nuclear receptor binding SET domain (NSD) family and SET-domain- containing 2 (SETD2) represent a new oncogene/tumor suppressor circuit controlling histone-H3 lysine-36 methylation (H3K36me). NSD1, NSD2, and/or NSD3 are histone methyltransferases that specify mono- and di-methylation of H3K36 (H3K36me1, H3K36me2). SETD2 is a histone methyltransferase that is specific for tri-methylation of H3K36 (H3K36me3); a methylation mark associated with active chromatin. Thus, in normal cells these proteins function in a stepwise manner to modify H3K36 to provide chromatin activation marks which serve as docking sites for transcriptional machinery and other H3K36me3 binding proteins to maintain chromatin structure. In cancer cells, the genes encoding both NSDs and SETD2 are frequently mutated. NSD mutations with gain of function (GOF) effects on H3K36me2 are found in acute lymphoblastic leukemia (ALL). We recently identified recurrent SETD2 mutations in both ALL and AML. We found that SETD2 mutations were associated with a dramatic reduction in the H3K36me3 mark and accumulation of H3K36me2 mark. Thus, we conclude that SETD2 genetic alterations lead to SETD2 loss of function (LOF), reduction of H3K36me3, and accumulation of H3K36me2. Based upon these novel, unique and exciting preliminary results, we hypothesize that although acute-leukemia-relevant mutations cause reciprocal biochemical effects in NSDs and SETD2, the end product of these changes is the same; a reduction in H3K36me3 and an accumulation of H3K36me2 (low H3K36me3/H3K36me2 ratio). Thus, in this application we will model and target novel NSD2 GOF mutations identified in leukemia. We will also model and target novel SETD2 LOF mutations identified in our leukemia screen. We will study their function on cell growth and leukemia development, on H3K36me3/H3K36me2 ratios and on H3K27 status. This proposed research should define functional relationships between NSDs and SETD2 in the context of leukemia, confirm the novel SETD2-H3K36me3 tumor suppressor pathway and identify novel complementary opportunities for therapeutic intervention on these epigenetic alterations.